Fig. 2. BCSC-secreted DKK1 shrinks the stem cell pool.
a Immunoblot assessment of β-CATENIN protein levels. Cells were cultured with the CM obtained from respective parental cells or mammosphere-enriched BCSCs and treated with DKK1 Inhibitor WAY262611 (1 μM) or vehicle. b Luciferase activities of the β-CATENIN reporter 8 × TOP Flash in MCF-7 cells cultured with the respective CM obtained from parental cells or mammosphere-enriched BCSCs, the β-CATENIN agonist CHIR99021 (1 μM) or inhibitor XAV939 (1 μM) served as the positive or negative control, respectively. c The gene expression profiles of pertinent WNT/β-CATENIN antagonists in matched pairs of ALDH+ and ALDH− MCF-7 or T47D cells. The data were downloaded from GEO GSE59653 or GSE80213, respectively. d Immunoblot assessment of DKK1 in the matched pairs of mammosphere-enriched BCSCs and parental cells. e ELISA quantification of secreted DKK1 levels in CM from paired mammosphere-enriched BCSCs or parental cells. f, g MCF-7 or T47D cells were cultured with the CM derived from respective parental cells or shCONT- or shDKK1-derived mammospheres for 48 h, the stemness properties were subsequently analyzed by ALDEFLUOR assay (f) or mammosphere-formation assay (g). Scale bars: 500 μm. h, i A series of limiting diluted luciferase-labeled MCF-7 cells were co-implanted with 4 × 105 unlabeled parental cells or shCONT- or shDKK1-derived mammosphere-enriched BCSCs into the second fat pad of host mice. BLI was performed on the tumor burden generated by MCF-7-luc cells (h), the CSC frequency was calculated using ELDA software (i). Results are shown as mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant (Unpaired two-tailed Student’s t test in (c, e), others one-way ANOVA followed by Tukey’s multiple comparison test). Source data are provided as a Source Data file.